Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Quercetin protects against Aspergillus fumigatus keratitis by reducing fungal load and inhibiting TLR-4 induced inflammatory response.

PURPOSE: To investigate the antifungal and anti-inflammatory effects of quercetin in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Draize eye test was performed in mice to evaluate the toxicity of quercetin, and the antifungal effects on A. fumigatus were assessed via scanning electron microscopy (SEM), propidium iodide uptake, and adherence assay. In fungal keratitis (FK) mouse models, immunostaining was performed for investigating toll-like receptor 4 (TLR-4) expression and macrophage infiltration. Real-time PCR, ELISA, and Western blot were used to evaluate the expression of pro-inflammatory factors IL-1ß, TNF-α, and IL-6 in infected RAW264.7 cells. Cells were also treated with TLR-4 siRNA or agonist CRX-527 to investigate mechanisms underlying the anti-inflammatory activity of quercetin. RESULTS: Quercetin at 32 µM was non-toxic to corneal epithelial and significantly inhibited A. fumigatus growth and adhesion, and also altered the structure and reduced the number of mycelia. Quercetin significantly reduced macrophage infiltration in the mouse cornea, and attenuated the expression of TLR-4 in the corneal epithelium and stroma of mice with keratitis caused by A. fumigatus. In RAW264.7 cells infected by A. fumigatus, quercetin downregulated TLR-4 along with pro-inflammatory factors IL-1ß, TNF-α, and IL-6. RAW cells with TLR-4 knockdown had reduced expression of factors after A. fumigatus infection, which was decreased even further with quercetin treatment. In contrast, cells with CRX-527 had elevated inflammatory factors compared to control, which was significantly attenuated in the presence of quercetin. CONCLUSION: Quercetin plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal load, disrupting hyphae structure, macrophage infiltration, and suppressing inflammation response in macrophages via TLR-4 mediated signaling pathway.

Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response.

Purpose: The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1. Results: In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1ß, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control. Conclusions: GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis.

PURPOSE: To explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. RESULTS: CS at 400 µg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1ß, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. CONCLUSION: CS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Lipoxin A4 alleviates inflammation in Aspergillus fumigatus-stimulated human corneal epithelial cells by Nrf2/HO-1 signaling pathway.

Purpose: To investigate the therapeutic effect of lipoxin A4 (LXA4) on Aspergillus fumigatus (A. fumigatus)-stimulated human corneal epithelial cells (HCECs). Methods: The cell counting kit-8 (CCK-8) was performed in HCECs to evaluate the toxicity of LXA4. A cell scratch test was used to assess the impact of LXA4 on the migration of HCECs. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were applied to examine the expression of inflammatory mediators in A. fumigatus-stimulated HCECs. The nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and expression in HCECs were detected by immunofluorescence staining. Results: LXA4 at 0-10 nmol·L-1 (nM) had no significant cytotoxic effect on HCECs. LXA4 at a concentration of 1 nM and 10 nM significantly promoted the migration rate of HCECs. The mRNA and protein levels of pro-inflammatory mediators, including IL-1ß, TNF-α, and IL-6, were remarkably lower in the LXA4-treated group. LXA4 promoted the expression of Nrf2 and heme oxygenase 1 (HO-1) in A. fumigatus-stimulated HCECs compared with the PBS control group. Pretreatment with brusatol (BT, Nrf2 inhibitor) or Zine Protoporphyrin (Znpp, HO-1 inhibitor) receded the anti-inflammatory ability of LXA4. Conclusions: LXA4 plays a protective role in A. fumigatus-stimulated HCECs by inhibiting the expression of pro-inflammatory mediators through the Nrf2/HO-1 signaling pathway.

Dysregulated Response of Follicular Helper T Cells to Hepatitis B Surface Antigen Promotes HBV Persistence in Mice and Associates With Outcomes of Patients.

BACKGROUND & AIMS: Production of neutralizing antibodies against hepatitis B surface antigen (HBsAg) is dysregulated in patients with persistent hepatitis B virus (HBV) infection. We investigated mechanisms by which this immune response to the virus is disrupted and whether it can be restored to promote clearance of HBV. METHODS: Immune-competent C57BL/6N and C57BL/6J, as well as mice deficient in follicular helper T cells (Tfh-cell-deficient), B cells, or Foxp3+ T-regulatory cells (Treg cell deficient), were given hydrodynamic injections of pAAV/HBV1.2 plasmids. Some mice were given injections of sorted Tfh cells, pan-B cells, Treg cells, or a blocking antibody against CTLA4. Production of antibodies against HBsAg and clearance of HBV were assessed by flow cytometry, enzyme-linked immunosorbent assay, polymerase chain reaction, and immunohistochemical analyses. We obtained blood samples from patients with HBV infection and isolated Treg cells. We measured the ability of Treg cells to suppress production of interleukin 21 (IL21) in CD4+ T cells. RESULTS: Immune-competent C57BL/6N and C57BL/6J mice transfected with the plasmid encoding HBV had features of viral clearance and viral persistence observed in humans. A Tfh-cell response to HBsAg was required for clearance of HBV and was suppressed by Treg cells in mice with persistent HBV infection. Depletion of Treg cells or inhibition of Treg-cell function (with blocking antibody against CTLA4) restored the Tfh-cell response against HBsAg and clearance of HBV in mice. Impaired Tfh-cell response to HBsAg was observed in blood from patients with chronic HBV infection, responsiveness was restored by depletion of Treg cells or blocking antibody against CTLA4. CONCLUSIONS: In studies of HBV-infected mice and blood from patients with chronic HBV infection, we found a Tfh-cell response to HBsAg of to be required for HBV clearance, and that this response was blocked by Treg cells. Inhibiting Treg-cell activity using neutralizing antibody against CTLA4 restored the ability of Tfh cells to clear HBV infection; this approach might be developed for treatment of patients with chronic HBV infection.